Effusion artifact: a chronic problem in DAB cytochemistry.
Effusion artifact: a chronic problem in DAB cytochemistry.
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渗出伪影：DAB 细胞化学中的一个长期问题。
DOI：
10.1177/28.12.7229340
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发表时间：
1980
期刊：
影响因子：
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通讯作者：
S. Goldfischer
中科院分区：
文献类型：
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作者：
S. Goldfischer
我同意 Novikoff 在两封给编辑的信中表达的担忧 (14, 16)，关于反应产物和酶或抗原的扩散、非反应位点的吸附以及组织保存不良引起的伪影，我想提请注意两个相关的 3, 3'-二氨基联苯胺 (DAB) 伪影。在看起来保存完好的组织中也可以看到细微的染色伪影。例如，一种可能未受到质疑的吸附伪影是在各种 DAB 培养基中孵育的组织中线粒体外膜的染色，这归因于细胞色素 b5 (1, 1 5)。这种染色仅在与反应性线粒体内膜相邻的小片段中可见（参见参考文献1中图9的插图），并且当仅嵴具有反应性时不存在（参见参考文献1中的图1 3）。对整个线粒体内膜进行染色可得到膜的复杂三维视图 (1 5)。内膜的两个反应性切割表面的图像通常不重叠，并且被误解为代表内膜和外膜染色（参考文献 17 中的图 8b）。除了酶和抗原的人为定位之外，细胞化学家还应该同样关注原始研究引用中的人为归因。例如，Novikoff 在他最近的信 (14) 中引用了 1972 年他自己实验室写给编辑的一封信 (16)，信中赞扬了 DAB，因为“其氧化产物是一种高度不溶的聚合物，在电子显微镜下观察时呈嗜锇性且均匀。”他自己的信（16）是这一基本观察的唯一参考文献。然而，所引用的信件和他实验室的任何其他出版物都没有提供任何证据证明氧化 DAB 的聚合性质。有关氧化 DAB 性质的证据来自其他研究人员。 Karnovsky (1 1) 在 1965 年的摘要中首次报道 DAB 反应产物“在四氧化锇中固定后为非结晶、不溶性且极不透明的电子。”第二年，Graham 和 Karnovsky (7) 将反应产物描述为“致密的、或多或少均质的物质……没有被 Epon 嵌入中使用的有机溶剂提取。”他们指出其化学性质未知，并提出：锇被氧化的 DAB 还原。氧化 DAB 是一种嗜锇聚合物的证据来自 Sehigman 等人 1968 年的报告。 (18) 诺维科夫在他最近的信 (14) 中没有引用这一点，并且在他 1972 年的信 (16) 中也没有提到这一点。
I share Novikoff’s concern, expressed in two Letters to the Editor (14, 16), regarding artifacts caused by diffusion of reaction product and enzyme or antigen, adsorption to nonreactive sites and poor preservation of tissue, and I would like to call attention to two related 3, 3’-diaminobenzidine (DAB) artifacts. Subtle staining artifacts can also be seen in tissues that appear to be very well preserved. For example, one likely adsorption artifact that has gone unchallenged is the staining ofouter mitochondrial membranes, which has been attributed to cytochrome b5, in tissues incubated in various DAB media ( 1 , 1 5). Such staining is seen only in small segments that are adjacent to reactive inner mitochondrial membrane (see inset to Figure 9 in ref. 1) and is not present when only the cristae are reactive (see Figure 1 3 in ref. 1 ). Staining of the entire inner mitochondrial membrane yields a complex three-dimensional view of the membrane ( 1 5 ). Images of the two reactive cut surfaces of the inner membrane usually do not overlap and have been misinterpreted as representing inner and outer membrane staining (Figure 8b in ref. 17). In addition to artifactual localization of enzymes and antigens, cytochemists should be equally concerned with artifactual attributions in the citation of original research. For example, in his recent Letter (14), Novikoff quotes from a 1972 Letter to the Editor from his own laboratory ( 16) that praises DAB because “the product ofits oxidation is a highly insoluble polymer that is osmiophihic and homogeneous when viewed by electron microscopy.” His own Letter (16) is the only reference given for this fundamental observation. However, neither the cited Letter nor any other publication from his laboratory has ever presented any evidence for the polymeric nature of oxidized DAB. The evidence concerning the nature of oxidized DAB came from other investigators. Karnovsky (1 1), in a 1965 abstract, first reported that DAB reaction product was “non-crystalline, insoluble, and extremehy electron opaque after fixation in osmium tetroxide. “ The following year Graham and Karnovsky (7) described the reaction products as a “dense, more or less homogeneous mass . . . not extracted by the organic solvents used in Epon embedding. “ They noted that its chemical nature was unknown and suggested that osmium was reduced by oxidized DAB. The evidence that oxidized DAB was an osmiophihic polymer came from a 1968 report by Sehigman et al. (18) that Novikoffdoes not cite in his recent Letter (14) and did not credit in this regard in his 1972 Letter (16).’